Linking sensory attributes to selected aroma compounds in South African Cabernet Sauvignon wines

wines

by

Emmanuelle Lapalus

Thesis presented in partial fulfilment of the requirements for the degree of

Master of Agricultural Science

at

Stellenbosch University

Department of Viticulture and Oenology, Faculty of AgriSciences

Supervisor: Prof Wessel Johannes du Toit

Declaration

By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification.

Date: _{March 2016}

Copyright © March 2016 Stellenbosch University All rights reserved

Summary

Cabernet Sauvignon is a widely planted red grape cultivar which produces worldwide, some of the finest and most expensive red wines. The typical aroma of Cabernet Sauvignon wines is often described as either fruity and berry-like or vegetative, herbaceous and green; the latter descriptors are often considered undesirable. High levels of 2-isobutyl-3-methoxypyrazine (ibMP), a powerful grape-derived compound, have been associated with greener notes in Cabernet Sauvignon wines. Over the past two decades, extensive research has been conducted worldwide to identify the active odorants that impact the aromatic profiles of

Cabernet Sauvignon wines. These compounds are mostly higher alcohols, esters, C13

-norisoprenoids, methoxypyrazines, sulphur compounds and certain terpenes. More recent studies have endeavoured to establish a relationship between the sensory analysis and chemical composition of these wines as it could help to explain the impact of certain odorants on the perception of either fruity or herbaceous notes. Despite the interest shown by the South African wine industry to improve the quality of Cabernet Sauvignon wines, no such study has been conducted in South Africa yet.

The first part of this study gives an overview of the major active aroma compounds which have been identified in Cabernet Sauvignon wines with a particular focus on volatile compounds that could exhibit either fruity berry notes or herbaceous/vegetative notes. Some of the findings of studies conducted in Australia and the United States are also discussed.

The second part of this study investigates the relationship between the volatile composition and sensory properties in 13 mono-varietal Cabernet Sauvignon wines produced in South Africa. The wines were selected to represent a broad range of fruity and herbaceous sensory attributes and were assessed by descriptive analysis. A limited number of volatile compounds (33 in total) that could contribute to either fruity or herbaceous characters, as indicated in the literature, were analysed using either headspace solid phase micro extraction (SPME) and gas chromatography–ion trap mass spectrometer detection (HS-SPME-GC-ion trap-MS analysis) or solid phase extraction (SPE) and gas chromatography coupled with a triple quadrupole detector (SPE-GC-MS/MS analysis). The statistical treatment by multiple factor analysis (MFA) of both compositional data and sensory data showed that certain volatile compounds such as

-damascenone, -ionone, dimethylsulphide (DMS) and ibMP predicted well some of the aroma

attributes used to describe the selected wines.

It was found that the analysis of 

-

dimethyl sulphide and ibMP could be of interest for winemakers wanting to explain certain typical aroma descriptors characterising South African Cabernet Sauvignon wines.

Opsomming

Cabernet Sauvignon is ‘n algemeen aangeplante rooidruif-kultivar wat wêreldwyd sommige van die beste and duurste rooiwyne produseer. Die tipiese aroma van Cabernet Sauvignon-wyne word gereeld as óf vrugtig en bessieagtig óf vegetatief, kruidagtig en groen beskryf; laasgenoemde beskrywende terme word in baie gevalle as ongewens beskou. Hoë vlakke van 2-isobutiel-3-metoksipirasien (ibMP), ‘n kragtige druifafgeleide verbinding, is reeds met die groener note in a Cabernet Sauvignon-wyne geassosieer. Oor die afgelope twee dekades is breedvoerige navorsing wêreldwyd onderneem om die aktiewe geurstowwe wat die aromatiese profiele van Cabernet Sauvignon-wyne beïnvloed, te identifiseer. Hierdie verbindings is meesal

hoër alkohole, esters, C13-norisoprenoïede, metoksipirasiene, swaelverbindings en sekere

terpene. Meer onlangse studies het gepoog om ‘n verhouding te bepaal tussen die sensoriese analise en chemiese samestelling van hierdie wyne, aangesien dit sou kon bydra tot die verklaring van die impak van sekere geurstowwe op die waarneming van óf vrugtige óf kruidagtige note. Ten spyte van die belangstelling wat deur die Suid-Afrikaanse wynbedryf daarin getoon is om die kwaliteit van Cabernet Sauvignon-wyne te verbeter, is geen sulke studies tot op hede in Suid-Afrika onderneem nie.

Die eerste deel van hierdie studie verskaf ‘n oorsig van die vernaamste aromaverbindings wat reeds in Cabernet Sauvignon-wyne geïdentifiseer is, met ‘n spesifieke fokus op vlugtige verbindings wat óf vrugtige bessienote of kruidagtige/vegetatiewe note kon vertoon. Sommige van die bevindings van studies wat in Australië en die VSA onderneem is, word ook bespreek.

Die tweede deel van hierdie studie ondersoek die verhouding tussen die vlugtige samestelling en sensoriese eienskappe in 13 enkelvariëteit Cabernet Sauvignon-wyne wat in Suid-Afrika geproduseer is. Die wyne is gekies om ‘n breë verskeidenheid van vrugtige en kruidagtige sensoriese eienskappe te verteenwoordig en is deur middel van beskrywende analise geassesseer. ‘n Beperkte aantal vlugtige verbindings (33 in totaal) wat óf tot die vrugtige óf die kruidagtige karakters kon bydrae, soos in die literatuur aangedui, is deur middel van óf lugspasie-analise (headspace solid phase micro extraction (SPME)) en gaschromatografie–ion trap massaspektrometrie waarneming (gas chromatogoraphy–ion trap mass spectrometer detection (HS-SPME-GC-ion trap-MS)) óf soliede fase ekstraksie (solid phase extraction (SPE)) en gaschromatografie tesame met ‘n a triple quadrupole detector (SPE-GC-MS/MS analise). Die statistiese behandeling deur veelvuldige faktor-analise (multiple factor analysis (MFA)) van beide die kompositoriese data en die sensoriese data het getoon dat sekere vlugtige verbindings, soos -damaskenoon, -ionoon, dimetielsulfied (DMS) en ibMP, sommige van die aroma-eienskappe wat gebruik word om die geselekteerde wyne te beskryf, goed voorspel het.

Daar is gevind dat die analise van 

-

dimetielsulfied en ibMP van belang kan wees vir wynmakers wat sekere van die tipiese aromabeskrywers wil verklaar wat Suid-Afrikaanse Cabernet Sauvignon-wyne karakteriseer.

Biographical sketch

Emmanuelle Lapalus was born in Chatillon sur Chalaronne France on 1 December 1971. She obtained a 3 year degree in Chemistry at the University of Dijon (France) in 1994 and obtained her HonsBSc-degree in Wine Biotechnology in 2012. She specialized in gas- and liquid chromatography and currently works as a GC/HPLC analyst at VinLAB Pty Ltd.

Acknowledgements

I wish to express my sincere gratitude and appreciation to the following persons and institutions:  Prof WJ du Toit (Department of Viticulture and Oenology, Stellenbosch University) who

acted as my supervisor, for his support, guidance and contribution to this study.

 Valeria Panzeri (Department of Viticulture and Oenology, Stellenbosch University) for her input with sensory and statistical analysis.

 Prof Martin Kidd (Centre for Statistical Consultation, Stellenbosch University) for his time and assistance with the processing and interpretation of statistical analysis.

 The winemakers who provided the wine samples for their interest in this study.  The management of VinLAB Pty Ltd for sponsoring my studies.

Preface

This thesis is presented as a compilation of four chapters.

Chapter 1 General Introduction and project aims

Chapter 2 Literature review

Linking volatile composition to sensory attributes in Cabernet Sauvignon wines

Chapter 3 Research results

Linking sensory attributes to selected aroma compounds in South African Cabernet Sauvignon wines

Table of Contents

Chapter 1. General Introduction and Project aims

1

1 Introduction 2

2 Project Aims 3

3 References 3

Chapter 2. Literature review

1 Introduction 7

2 Volatile compounds contributing to fruity, berry-like aromas 8

2.1 Higher alcohols 8

2.2 Esters 9

2.3 C13-norisoprenoids 11

2.4 Sulphur compounds 12

2.4.1. Reductive sulphur compounds 12

2.4.2. Varietal thiols 13

3 Volatile compounds contributing to herbaceous/vegetative characters 15

3.1 Methoxypyrazines 15

3.2 C6 and C9 derivatives: Green leaf volatiles 17

3.3 Monoterpenes 18

4 Chemical composition and sensory analysis of Cabernet Sauvignon wines 19

5 Conclusion 22

6 References 23

Chapter 3. Research results

29

1 Introduction 30

2 Materials and methods 31

2.1 Wines 31

2.2 Descriptive analysis 33

2.3 Chemical analyses 35

2.3.1 Conventional oenological parameters 35

2.3.2 Volatile compounds 35

2.3.2.1 Reagents, standards and material 35

2.3.2.2 General analysis of wine volatiles 36

2.3.2.3 Methoxypyrazines analysis 37

2.3.2.4 Dimethyl sulphide analysis 38

2.3.2.5 Volatile thiols 39

2.4 Statistical analysis of data 40

2.4.1 Descriptive analysis 40

2.4.2 Linking chemical and Descriptive Analysis data 40

3.1 Descriptive Analysis 40

3.2 Chemical analyses 46

3.2.1 Conventional oenological parameters 46

3.2.2 Volatile compounds 47

3.3 Correlation between descriptive analysis and chemical data 50

3.3.1 Results 50

3.3.2 Discussion 55

4 Conclusion 57

5 References 58

Addendums 60

Chapter 4. General discussion and conclusion

64

1 Conclusion and future prospects 65

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Introduction and

project aims

1 Introduction

Vitis vinifera L. cv. Cabernet Sauvignon cultivar is internationally known for the prestigious wines produced from it in the Bordeaux region of France where it originates. Cabernet Sauvignon vines have the ability to grow in a variety of climates and soil types (Carey et al., 2008) which explains why Cabernet Sauvignon has become a popular, widespread red grape variety in many other wine producing countries including Australia, the United States of America, South Africa and recently China (Carey et al., 2008; Tao & Li, 2009; Robinson et al., 2011, Hjelmeland et al., 2013). Although Cabernet Sauvignon is a versatile grape cultivar which can thrive in various climatic conditions, it performs at its best in warm regions with well-drained soils. (Roujou de Boubée et al., 2000; Oberholster et al., 2010). High quality grapes produce tannic wines with an intense dark red colour which often exhibits red berry, black berry and spicy aromas (Oberholster et al., 2010). In cooler climates, Cabernet Sauvignon wines tend to develop greener notes described as green pepper, mint and cut grass which are perceived as a lack of ripeness and can be detrimental to their quality (Allen et al., 1994; Allen & Lacey, 1998; Roujou de Boubée et al., 2000). The overall perceived aroma of wines derives directly or indirectly from the grape composition at the time of harvest (Carey et al., 2008; Polášková et al., 2008). Thus, a great deal is done in the vineyard so that the grapes reach optimum maturity, translating into the optimal chemical composition (including colour, sugar levels and amino acids) at the time of harvest to produce Cabernet Sauvignon wines that are fruitier and still present an intense darker colour (Oberholster et al., 2010).

Worldwide, the sensory evaluations conducted on Cabernet Sauvignon wines have often led to two different sets of descriptors: one characterised by fruity, berry notes and the other by vegetative/herbaceous notes (Heymann & Noble, 1987; Chapman et al., 2005; Robinson et al.; 2011). Moreover, gas chromatography-olfactometry (GC-O) techniques have helped to identify important impact odorants of Cabernet Sauvignon wines (Lopez et al., 1999; Kotseridis & Baumes, 2000; Gȕrbȕz et al., 2006; Falcao et al., 2008). The volatile compounds that contribute to the fruity notes are ethyl esters, 2-phenyl ethanol, -ionone, -damascenone and 3-mercaptohexan-1-ol (Kotseridis & Baumes, 2000). The vegetative, herbaceous notes described as green pepper, cut grass and mint have been attributed to methoxypyrazines and especially 2-isobutyl-3-methoxypyrazine (ibMP) (Allen et al., 1994; Allen & Lacey, 1998; Roujou de

Boubée et al., 2000), but also to some aldehydes and alcohols C6 and C9 derivatives such as

hexanol, cis-3-hexenol and nona-2,6-dienal (Kotseridis & Baumes, 2000; Kalua & Boss, 2009; Callejón et al., 2012) and certain monoterpenes such as 1,8-cineole (Capone et al., 2011).

Recent studies conducted in Australia and the United States of America have investigated the relationship between sensory attributes and wine composition in Cabernet Sauvignon wines. The authors were particularly interested in the fruity/berry notes and the herbaceous/vegetative notes and how they linked to the chemical composition of these wines (Robinson et al.; 2011; Hjelmeland et al., 2013; Bindon et al., 2014). Despite the fact that Cabernet Sauvignon is one of

the most planted red grape variety in South Africa (SAWIS, 2014), grown to produce some of its most expensive and iconic wines, such research has not yet been conducted in South Africa. Little has been published on the perceptual aromatic properties and chemical composition of South African Cabernet Sauvignon wines. A better knowledge thereof could benefit the wine industry and help to produce Cabernet Sauvignon wines with more desirable perceptual properties.

2 Project

aims

The aim of this study was mainly to investigate the relationship between the sensory attributes and the volatile composition of a selected number of South African Cabernet Sauvignon wines. The specific aims were as follows:

(i) select single mono varietal Cabernet Sauvignon wines that exhibit a broad range

of herbaceous or fruity notes,

(ii) characterise the aroma profiles of the selected wines by descriptive analysis,

(iii) select previously reported aroma-active components, arising mostly from grape

composition and yeast metabolism that are responsible for, either the fruity notes or the herbaceous/vegetative notes,

(iv) analyse and quantify the selected aroma compounds in the different wines using

gas-chromatography, and

(v) investigate the relationship between sensory attributes and selected chemical

compounds.

3 References

Allen, M. S., Lacey, M. J. & Boyd, S. (1994). Determination of methoxypyrazines in red wines by stable isotope dilution gas-chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry. 42, 1734-1738.

Allen, M. S. & Lacey, M. J. (1998). Methoxypyrazines of grapes and wines In: Waterhouse, A. L. & Ebeler, S. E. (eds), Chemistry of wine flavour. American Chemical Society. Washington DC. pp 31-38.

Bindon, K., Holt, H., Varela, C., Williamson, P. O., Herderich, M. & Francis, I. L. (2014). Relationships between harvest time and wine composition in Vitis vinifera L. cv. Cabernet Sauvignon. 2. Wine sensory properties and consumer preference. Food Chemistry. 154, 90-101.

Callejón, R. M., Margulies, B., Hirson, G. D. & Ebeler, S. E. (2012). Dynamic changes in volatile compounds during fermentation of Cabernet Sauvignon grapes with or without skins. American Journal of Enology and Viticulture. 63, 301-312.

Capone, D. L., Van Leeuwen, K., Taylor, D. K., Jeffery, D. W., Pardon, K. H., Elsey, G. M. & Sefton, M. A. (2011). Evolution and occurrence of 1,8-cineole (eucalyptol) in Australian wine. Journal of Agricultural and Food Chemistry. 59, 953-959.

Carey, V. A., Archer, E., Barbeau, G. & Saayman, D. (2008). Viticultural terroirs in Stellenbosch, South Africa. II. The interaction of Cabernet Sauvignon and Sauvignon Blanc with environment. Journal International des Sciences de la Vigne et du Vin. 42, 185-201.

Chapman, D. M., Roby, G., Ebeler, S. E., Guinard, J-X. & Matthews, M. A. (2005). Sensory attributes of Cabernet Sauvignon wines made from vines with different water status. Australian Journal of Grape and Wine Research. 11, 339-347.

Falcao, L. D., De Revel, G., Rosier, J-P. & Bordignon-Luiz, M.T. (2008). Aroma impact components of Brazilian Cabernet Sauvignon wines using detection frequency analysis (GC-olfactometry). Food Chemistry. 107, 497-505.

Gȕrbȕz, O., Rouseff, J. M. & Rouseff, R. L. (2006). Comparison of aroma volatiles in commercial Merlot and Cabernet Sauvignon wines using gas chromatography-olfactometry and gas chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry. 54, 3990-3996.

Heymann, H. & Noble, A. C. (1987). Descriptive analysis of commercial Cabernet Sauvignon Wines from California. American Journal of Enology and Viticulture. 38, 41-44.

Hjelmeland, A.K., King, E. S., Ebeler, S. E. & Heymann, H. (2013). Characterizing the chemical and sensory profiles of United States Cabernet Sauvignon wines and blends. American Journal of Enology and Viticulture. 64, 169-179.

Kalua, C. M. & Boss, P. K. (2009). Evolution of volatile compounds during the development of Cabernet Sauvignon grapes (Vitis vinifera L.). Journal of Agricultural and Food Chemistry. 57, 3818-3830.

Kotseridis, Y. & Baumes, R. (2000). Identification of impact odorants in Bordeaux red grape juice, in the commercial yeast used for its fermentation and in the produced wine. Journal of Agricultural and Food Chemistry. 48, 400-406.

Lopez, R., Ferreira, V., Hernandez, P. & Cacho, J. F. (1999). Identification of impact odorants of young red wines made with Merlot, Cabernet Sauvignon and Grenache grape varieties: A comparative study. Journal of the Science of Food and Agriculture. 79, 1461-1467.

Oberholster, A., Botes, M-P. & Lambrechts, M. (2010). Phenolic composition of Cabernet Sauvignon (Vitis vinifera) grapes during ripening in four South African winegrowing regions. Journal International des Sciences de la Vigne et du Vin. Special issue Macrowine, June 2010, 33-40.

Polášková, P., Herszage, J. & Ebeler, S. (2008). Wine flavor: chemistry in a glass. Chemical Society Reviews. 37, 2478-2489.

Robinson, A. L., Adams, D. O., Boss, P. K., Heymann, H., Solomon, P. S. & Trengrove, R. D. (2011). The relationship between sensory attributes and wine composition for Australian Cabernet Sauvignon wines. Australian Journal of Grape and Wine Research. 17, 327-340.

Roujou de Boubée, D., Van Leeuwen, C. & Dubourdieu, D. (2000). Organoleptic impact of 2-methoxy-3-isobutylpyrazine on red Bordeaux and Loire wines. Effect of environmental conditions on concentrations in grapes during ripening. Journal of Agricultural and Food Chemistry. 48, 4830-4834.

SAWIS (2014). South African Wine Industry Information and Systems - Status of wine grape

Vines as on 31 December 2013. (WWW document). URL http://www.sawis.co.za/info/.

March 2014.

Tao, Y-S. & Li, H. (2009). Active volatiles of Cabernet Sauvignon wine from Changli County. Natural Science. 1, 176-182.

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Literature review

Linking volatile composition to sensory attributes in

Cabernet Sauvignon wines

1 Introduction

Wine aroma is the result of a complex mixture of chemical compounds derived from grapes, yeast and bacterial metabolism during vinification and, if used, oak wood during barrel ageing (Francis & Newton, 2005). Grape quality at the time of harvest is the foundation for the production of quality wines (Oberholster et al., 2010). The grape berry provides most of the substrates needed for the yeast and lactic acid bacteria to function: sugars, fatty acids, nitrogen and sulphur-containing compounds are metabolised into volatile compounds (Callejón et al., 2012). Grapes also contain odourless precursors that can be released by the yeast during fermentation. Grape composition depends on the grape variety (Hernandez-Ortez et al., 2002) and environmental and viticultural conditions, which include the type of soil, pruning and training systems, density of plantation, etcetera, all of which can have a strong influence on grape composition at véraison and on variations in ripening (Robinson et al., 2014).

Worldwide, Cabernet Sauvignon has become very popular and often produces some of the most expensive wines (Tao & Li, 2009; Robinson et al.; 2011, Hjelmeland et al., 2013). Cabernet Sauvignon is the most widely planted red grape in the United States (Hjelmeland et al., 2013), it was ranked the third most planted grape variety in Australia in 2009 (Robinson et al., 2011) and accounted for 72% of the total grape-producing areas in China (Tao & Li, 2009). In 2013, 11.7% of the area under vines in South Africa was planted to Cabernet Sauvignon, making it the predominant red cultivar in the country (SAWIS, 2014).

Cabernet Sauvignon wines are often characterised by two antagonistic aromatic profiles: one with fruity, berry-like aromas and the other with vegetative, herbaceous aromas (Heymann & Noble, 1987; Chapman et al., 2005; Carey et al., 2008; Preston et al., 2008; Robinson et al., 2011; Bindon et al., 2013a). Worldwide, studies have been conducted to establish relationships between the sensory attributes and chemical composition of Cabernet Sauvignon wines (Kotseridis & Baumes, 2000; Ferreira et al., 2000; Falcao et al., 2008; Escudero et al., 2007; Robinson et al., 2011; Forde et al., 2011).

The active odorants that have been characterised in these studies belong to different

chemical groups, consisting mostly of higher alcohols, esters, C13-norisoprenoids,

methoxypyrazines, sulphur compounds, aldehydes and terpenes. This literature review will thus focus on the prevalent volatile aroma compounds that have been characterised in Cabernet Sauvignon wines and how they relate to the sensory composition of the wines.

2 Volatile compounds contributing to fruity, berry-like aromas.

Cabernet Sauvignon wines are often described as exhibiting fruity aromas, such as fresh cherry, red or black berry, jam/cooked berry, cooked fruit, dried fruit and raisin (Heymann & Noble, 1987; Chapman et al., 2005). The compounds that have been associated, directly or indirectly,

with the fruity aromas in Cabernet Sauvignon wines are mostly higher alcohols, esters, C13

-norisoprenoids and sulphur compounds.

2.1 Higher alcohols

Higher alcohols are alcohols containing more than two carbon atoms. They are quantitatively one of the most important groups of secondary metabolites formed during alcoholic fermentation. Their concentrations in wine range from less than 100 mg/l to 300 mg/l and above, with white wines generally exhibiting the lowest levels. Levels below 300 mg/l contribute positively to the aromatic profile of the wine, while higher levels impact negatively (Ugliano & Henschke, 2009).

Sugar levels, yeast strain, aeration and fermentation temperature are factors to consider in the production of higher alcohols, but the amino acid composition of the must certainly plays the most important role. Moreover, each grape variety presents a relatively characteristic amino acid profile that will determine the eventual volatile composition of the wine (Hernandez-Ortez et al., 2002). Higher alcohols are formed from two intertwined pathways that produce α-keto acids as intermediates for the degradation of amino acids (Ehrlich reaction) or their biosynthesis from glucose (anabolic pathway). The availability or deficiency of amino acids in the must determines which pathway will be used during yeast growth (Lambrechts & Pretorius, 2000; Ugliano & Henschke, 2009). Higher alcohols are constituted of either aliphatic or aromatic alcohols, with propanol, isobutanol, isoamyl alcohol and 2-phenylethanol being the major congeners found in wine.

Two higher alcohols that have been characterised as active odorants in Cabernet Sauvignon wines in a number of studies are 2-phenylethanol (or phenethyl alcohol) and 3-methyl-1-butanol (Lopez et al., 1999; Ferreira et al., 2000; Kotseridis et al., 2000; Falcao et al., 2008). 2-phenylethanol has a honey, rose-like aroma and plays an important role in Cabernet Sauvignon wines’ aromatic properties when found at above threshold levels (Falcao et al., 2008). 2-phenylethanol is produced from phenylalanine by the Ehrlich reaction and its concentration depends on the yeast strain. Higher pH and fermentations at 15 or 25°C rather than 35°C yield higher levels (Rankine & Pocock, 1969). Table 1 lists the major higher alcohols, their sensory thresholds and concentrations that have been detected in Cabernet Sauvignon wines.

Table 1: Main higher alcohol congeners, their sensory thresholds and concentrations found in

Cabernet Sauvignon wines.

Higher alcohols Aroma Odour threshold (µg/l)

Concentrations in Cabernet Sauvignon wines (µg/l)

1-propanol Ripe fruit 50 0001a 5 824-20 3954

isobutanol Solvent-like 40 0002a 31 005-105 2124 1-butanol Powerful, fresh,

green grass odour 150 000

1a _{1 556-4 712}4 Isoamyl alcohol (3-methyl-1-butanol) Whiskey, malt, marzipan 30 000 2a _{164 391-567 524}4 _{/ 179 000-205 000}5 2-phenylethanol

(Phenethyl alcohol) Honey, rose 14 000

3b _{30 783-140 086}4 _{/ 42 730-90 160}5

1 Cullere et al., 2004; 2 Guth 1997; 3 Ferreira et al., 2000; 4 Tao & Zhang, 2010; 5 Bindon et al., 2013b a In water/ethanol (90+10,w/w); b In model wine

2.2 Esters

Some volatile esters are synthesised in Cabernet Sauvignon grapes throughout the stages of berry development, but their contribution to wine aroma is not significant (Kalua & Boss, 2009). Esters found in Cabernet Sauvignon wines are mostly secondary metabolites of the fermentation. Although volatile esters are present at lower concentrations compared to higher alcohols, they have a greater impact on the wine aroma due to their lower odour thresholds.

Esters are divided into two groups: acetic esters of higher alcohols and ethyl esters of fatty acids. Acetate esters are the result of the reaction of acetyl-CoA with higher alcohols. The higher alcohols are formed from the degradation of amino acids, while hexanol is formed through the lipoxygenase pathway activated at crushing (Joslin & Ough, 1978). Acetate esters have intense fruity aromas: isoamyl acetate has an aroma of banana and hexyl acetate has an aroma reminiscent of apple. Fatty acid ethyl esters are the products of the ethanolysis of the acyl-CoA formed during fatty acid synthesis (ethyl lactate occurs after malolactic fermentation from the formation of lactic acid) or degradation. Both groups of esters contribute to the fruitiness of wine aroma (Ugliano & Henschke, 2009).

Branched ethyl esters, such as ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, ethyl 2-, 3-, 4-methypentanoate, and one cyclic ester, namely ethyl cyclohexanoate, have recently been identified as being important contributors to the sweet-fruity aroma in wine (Escudero et al., 2007; Pineau et al., 2009). Recent studies show that the fruity aroma in wine arises from a collective contribution, rather than individual contributions by esters. In one study, the berry fruity notes of red wines were related to the addition effect of nine fruity esters (Escudero et al., 2007). Pineau et al. (2009) reported that blackberry aromas in red wines were associated with higher than average levels of ethyl propanoate, ethyl methylpropanoate and ethyl 2-methylbutanoate while red berry aromas were associated with ethyl butanoate, ethyl hexanoate, ethyl octanoate and ethyl 3-hydroxybutanoate.

Concentrations of esters are dependent on grape variety, grape maturity, yeast strain, temperature of fermentation and the juice amino acid content (Ugliano & Henschke, 2009). Enzymatic hydrolysis of esters occurs during fermentation and chemical hydrolysis occurs during storage and ageing (Lambrechts & Pretorius, 2000). The esters having an important impact on the aromatic properties of Cabernet Sauvignon wines are listed in Table 2.

Table 2: Important esters found in Cabernet Sauvignon wines and their odour thresholds.

Esters Aroma Odour

threshold (µg/l)

Concentrations reported in Cabernet Sauvignon wines

(µg/l)

Ethyl esters of fatty acids

Ethyl butyrate Papaya, apple 6001a _{530–1 941}4

Ethyl hexanoate Green apple 4401a _{373–1 315}4

Ethyl octanoate Pear 9601a 125–7414

Ethyl decanoate Grape 2002b 203-4021 / 4–1094 Ethyl lactate Raspberry 154 0003c 43 284–237 4154 Ethyl 2-methylbutanoate Fruity 56001a 162-4261 / 9.2-323 Ethyl 3-methylbutanoate Fruity, anise 32b 20-253/14,4–27,35

Acetate esters

Isobutyl acetate Solvent 21001a _70-1804

Isoamyl acetate Banana 18301a _{205–2 784}4

Phenethyl acetate Roses 2502b _83–4904 _{/ 70–165}5

Hexyl acetate Pear 15001a _7–194 _{/ 57–77}5

1 Pineau et al., 2009, 2 Ferreira et al., 2000, 3 Escudero et al., 2007, 4 Tao & Zhang, 2010; 5 Bindon et al., 2013b a In dearomatized red wine b In a synthetic wine; c In wine

2.3 C13-norisoprenoids

C13-norisoprenoids are grape-derived compounds that are formed from the degradation of

carotenoids. They are present in the berry as non-volatile, non-odorant glycosidic compounds (Baumes et al., 2002). Marais et al. (1999) showed that light exposure and leaf removal

increase the concentration of C13-norisoprenoids in the grapes. Carotenoids are synthesised

between berry set and véraison, then degrade from véraison to maturity, producing C13

-glycosylated norisoprenoids. Due to their low odour thresholds, the C13-norisoprenoids are

among the most potent aromatic components found in wine and contribute greatly to floral and fruity notes in red and white wines (Schwab et al., 2008).

-damascenone and -ionone are two major C13-norisoprenoids found in wine.

-damascenone has an apple, rose and honey aroma, while -ionone has a seaweed, violet and raspberry aroma (Francis & Newton, 2005). Both are stored in the berry as odourless glycosylated precursors and are released under the acidic conditions of the must and through fermentation.

At maturity, C13-norisoprenoids are more abundant in berries exposed to sunshine than in

shaded berries (Baumes et al., 2002). -damascenone is the result of the degradation of neoxanthine, and -ionone is a secondary metabolite of the degradation of -carotene.

The analysis of -ionone in red wines from the Bordeaux region showed that it is an important impact odorant. Its levels in the berry tend to decrease during ripening, but the levels found in wine are higher than or near to its odour threshold estimated at 90 ng/l in a model wine (Kotseridis et al., 1999b). Reported levels of -ionone in Cabernet Sauvignon wines vary from 0.08 to 0.37 µg/l (Kotseridis et al., 1999b; Falcao et al., 2008).

-damascenone has been identified as being an active odorant in Cabernet Sauvignon wines in many studies (Kotseridis et al., 1999c; Lopez et al., 1999; Falcao et al., 2008; Tao & Li, 2009). Higher levels of -damascenone impart peach or canned apple notes which positively benefit the aromatic properties of Cabernet Sauvignon wines (Ferreira et al., 2000; Falcao et al., 2008). According to Pineau et al. (2007) -damascenone mostly acts as an enhancer of red fruit aroma and its odour threshold in red wine probably ranges from 2 to 7 µg/l. Reported levels of -damascenone in Cabernet Sauvignon wines vary from 1.25 to 17.7 µg/l (Pineau et al., 2007; Falcao et al., 2008; Bindon et al., 2013b).

2.4 Sulphur compounds

Sulphur compounds are present in wine as sulphides, polysulphides, heterocyclic compounds, thioesters and thiols. They have low odour thresholds (from low ppt to low ppb levels) and thus account for some of the most potent odorants in wines (Mestres et al., 2000).

Sulphur compounds originate from two main processes that are either enzymatic (degradation of sulphur-containing amino acids, formation of fermentation products and metabolism of sulphur-containing pesticides) or non-enzymatic (photochemical, thermal and other reactions during winemaking and storage) (Mestres et al., 2000). Sulphur compounds present different olfactory qualities: some sulphur-containing compounds cause reductive aroma characters ranging from onion to cabbage and burnt rubber, while others, like 4-mercapto-4-methylpentan-2-one, 3-mercaptohexan-1-ol and 3-mercaptohexyl acetate, are impact odorants contributing to the varietal characteristics of certain wines (Mestres et al., 2000; Coetzee & Du Toit, 2012).

2.4.1 Reductive sulphur compounds

Reduction in wine is associated with sulphur compounds having aromas reminiscent of rotten egg, cabbage, onion, garlic and burnt rubber. The main volatile sulphur compounds responsible

for these off-odours include H2S, methanethiol, ethanethiol, dimethyl sulphide, and other

sulphides and disulphides (Park et al., 1994; Mestres et al., 2000). H2S acts as an intermediate

product in the sulphate reduction sequence (SRS) pathway, which is activated to feed the metabolic demand for cysteine and methionine, two sulphur-containing amino acids. The yeast cell utilises sulphate and sulphite readily present in must to synthesise sulphur-containing amino acids. When there is a deficiency of nitrogen and precursors of sulphur amino acids

(O-acetylhomoserine and O-acetylserine), H2S is no longer metabolised by the yeast cell and it

starts accumulating in the must. During and after fermentation, H2S reacts with ethanol and

methanol to form the corresponding mercaptans: methanethiol and ethanethiol (Swiegers & Pretorius, 2007).

Dimethyl sulphide (DMS) is another low molecular weight sulphur compound linked to reduction in wine. To date, the pathways leading to the production of dimethyl sulphide in wine have not been elucidated fully. It is thought to be formed by the yeast during fermentation from sulphur-containing amino acids such as cysteine, cystine and glutathione. Cysteine supplements in a culture medium subjected to fermentation by Saccharomyces cerevisiae led to the production of DMS (De Mora et al., 1986). The levels of DMS found in young wines are usually low and below its perception threshold, which is 27 µg/l in red wines (Segurel et al., 2004). Levels of DMS increase during ageing and wine storage as a result of the degradation of dimethyl sulphoxide (DMSO) and of S-methyl-L-methionine (Swiegers et al., 2005). In storage experiments, bottled wines that had been spiked with DMSO and cysteine presented increased levels of DMS, indicating that DMSO is a potential precursor of DMS during bottle ageing (De

Mora et al., 1993). In a survey screening 77 Californian wines, dimethyl sulphide (DMS) was found to be the most widely distributed and most abundant sulphur-containing compound (Park et al., 1994). DMS has an aroma reminiscent of asparagus, cooked corn and molasses (Swiegers et al., 2005). However, some authors have reported that low levels of DMS exhibit herbaceous, vegetal and quince-like aromas (Mestres et al., 2000) and have the ability to enhance the fruity notes of red wines (Segurel et al., 2004; Escudero et al., 2007).

2.4.2 Varietal thiols

Varietal thiols are a group of sulphur compounds with extremely low odour thresholds, accounting for some of the most powerful aroma notes found in wine. 4-mercapto-4-methyl-pentan-2-one (4MMP), 3-mercaptohexyl acetate (3MHA) and 3-mercaptohexan-1-ol (3MH) have been identified as three major aroma compounds contributing to the varietal aroma of Sauvignon Blanc wines (Darriet et al., 1995; Tominaga et al., 1998a).

Bouchilloux et al. (1998) identified 3MH and 3MHA in the Bordeaux red wine varieties

Merlot and Cabernet Sauvignon, and noted that the aromatic complexity of a Cabernet Sauvignon or Merlot wine was significantly decreased by the simple addition of copper sulphate. These compounds exhibit powerful aromas, ranging from black currant bud (4MMP) to grapefruit (3MH) and passion fruit and box tree (3MHA) (Roland et al., 2011).

4MMP and 3MH are almost non-existent in grape juice and are released during fermentation from odourless non-volatile precursors synthesised in the grape berry (Dubourdieu et al., 2006). 3MHA arises from the acetylation of 3MH during fermentation by the action of the yeast ester, forming alcohol acetyltransferase (Roland et al., 2011). Three metabolic pathways leading to the production of 4MMP and 3MH have been identified (Roland et al., 2011). Two of these pathways are shared by 4MMP and 3MH and involve cysteinylated and glutathionylated precursors (Tominaga et al., 1998a; Peyrot des Gachons et al., 2002). The third pathway

leading to the formation of 3MH involves trans-2-hexenal as well as trans-2-hexenol, with H2S

as a sulphur donor (Harsch et al., 2013). Experimental trials show that a delay in the metabolisation of both trans-2-hexenol and trans-2-hexenal, combined with sufficient levels of

H2S, could significantly increase the production of 3MH. However, these conditions are not

easily met in commercial fermentations, as H2S would have to be produced timeously to react

with both C6 derivatives before they are metabolised in the early stages of fermentation (Harsch

et al., 2013).

The levels of precursors formed in the berry depend on a number of parameters, such as soil, microclimate, maturity and operations prior to fermentation (Murat et al., 2001a), and only a small percentage of the precursors available in the must are released as volatile thiols during fermentation (Murat et al., 2001b; Dubourdieu et al., 2006). The yeast strains and species play a decisive role in the levels of 4MMP and 3MH released in wine (Murat et al., 2001a; Howell et al., 2004; Dubourdieu et al., 2006), as well as in the conversion of 3MH into 3MHA. The

temperature of fermentation also has a strong effect on the production of 4MMP; some strains are able to release up to 100-fold more 4MMP than others when fermenting at 28°C rather than at 18°C (Howell et al., 2004). The cysteinylated precursor of 3MH is mainly located in the skins of Merlot and Cabernet Sauvignon grapes (Murat et al., 2001b), and the cysteinylated precursor of 4MMP is present in both the skin and the pulp (Peyrot des Gachons et al., 2002). Prolonged juice-skin contact increases the content of the precursor in the must, and this is even more so at higher temperatures (Murat et al., 2001b). The thiols are released during fermentation, probably from a β-lyase activity of Saccharomyces cerevisiae (Peyrot des Gachons et al., 2000).

Table 3 lists some sulphur compounds that have an aromatic impact on Cabernet Sauvignon wines, along with their sensory thresholds and concentrations.

Table 3: Sulphur compounds having a positive aromatic impact on Cabernet Sauvignon wines.

Sulphur compounds Aroma Odour threshold

(µg/l)

Concentrations reported in

Cabernet Sauvignon wines dimethylsulphide Asparagus, corn molasses

herbaceous 10–1601a 602b 2–5,34 5–605 4-mercapto-4-methylpentan-2-one

Box tree, guava, black

currant 0,003

3c _–

3-mercaptohexanol Grapefruit, passion fruit 0,0603c 10–5 0006 3-mercaptohexyl acetate Box tree, passion fruit 0,0043c 1–2006

1 Mestres et al., 2000; 2 Swiegers et al., 2005; 3 Tominaga et al., 1998b; 4 Bindon et al., 2013b; 5 Park et al., 1994; 6 Bouchilloux et al., 1998 (approximate values)

3

Volatile compounds contributing to herbaceous, vegetative aromas

The vegetative/herbaceous character of Cabernet Sauvignon wines encompasses a number of attributes/descriptors, such as bell pepper, fresh green, fresh, cool, minty and cooked asparagus (Roujou de Boubée et al., 2000; Capone et al., 2011; Bindon et al., 2014). The compounds associated with these descriptors commonly belong to the following chemical

groups: methoxypyrazines, C6 alcohols and aldehyde derivatives and certain monoterpenes.

The ambivalent role of DMS, which can contribute to the fruity and vegetative/herbaceous character of red wines, was discussed earlier.

3.1 Methoxypyrazines

Methoxypyrazines are a class of compounds that contribute to the varietal character of Sauvignon Blanc, Semillon, Cabernet Sauvignon and Merlot and impart a herbaceous, vegetal or green aroma (Allen & Lacey, 1998). A comprehensive study of 29 different grape cultivars showed that Cabernet Sauvignon, Merlot, Cabernet franc, Sauvignon blanc and Semillon are the only cultivars presenting significant, measurable levels of 2-isobutyl-3-methoxypyrazine (ibMP) from pre-véraison to harvest. The fact that ibMP only occurs in some cultivars points towards a genetically programmed trait of closely related cultivars (Koch et al., 2010). Three principal methoxypyrazines with low odour thresholds contribute the most to wine aroma. These are 2-isobutyl-3-methoxypyrazine (ibMP), 2-isopropyl-3-methoxypyrazine (ipMP) and 2-sec-butyl-3-methoxypyrazine (sbMP), a less important compound (Table 4). While sbMP and ipMP are mostly present in wine at levels nearing their perception thresholds, ibMP is often found at higher, above-thresholds concentrations (Allen et al., 1994; Roujou de Boubée et al., 2000). ibMP is a potent aroma-active compound: low levels contribute to the aromatic complexity of red wines, but higher levels are perceived as a lack of ripeness and are detrimental to wine quality (Allen & Lacey, 1998; Roujou de Boubée et al., 2000). The recognition threshold of ibMP in red wine was established at 15 ng/l (Roujou de Boubée et al., 2000).

The levels of ibMP in the berry decrease during fruit maturation (Roujou de Boubée et al., 2002; Ryona et al., 2008; Scheiner et al., 2012). Kotseridis et al., (1999a) observed a 50% decrease in ibMP concentration, with a 15-day delay in harvesting. During the ripening of Cabernet Sauvignon grape bunches, ibMP is found mostly in the stems (53.4%), skin (31%) and seeds (15%), and the levels of ibMP in the skins increases from pre-véraison to harvest, reaching 95.5% of the total ibMP levels, while the levels in the stems and seeds decrease (Roujou de Boubée et al., 2002). Some studies have reported concomitant decreases in malic acid and ibMP levels during ripening, suggesting that monitoring the malic acid levels in the berry could be a good indicator of ibMP levels at harvest (Kotseridis et al., 1999a; Roujou de Boubée et al., 2000). Ryona et al. (2008), however, found that malic acid and ibMP levels are not always well correlated.

Marais et al. (1999) investigated the effect of average temperature and solar radiation within the canopies of Sauvignon blanc vines in three climatically different South African regions (Stellenbosch, Robertson and Elgin). Some of the vines were manipulated (trained and defruited) in a way that increased shading of the grape clusters, and these were then compared to control vines that were not manipulated. The recording of the temperatures within the canopy and within the clusters, as well as the recording of solar radiation within the canopies, gave an indication of the microclimatic conditions in the vines. In the end, it was found that higher ibMP levels were correlated with cooler seasons and regions.

ibMP levels accumulate in the berry from fruit set to about two to three weeks before véraison, from there on the levels decreased until harvest and it appears that pre-véraison cluster light exposure has a critical impact on ibMP levels at harvest (Roujou de Boubée et al., 2002; Ryona et al., 2008). Roujou de Boubée et al. (2002) reported a significant decrease in ibMP levels (68.4%) at harvest as a result of pre-véraison cluster light exposure. In a recent study, Ryona et al. (2008) compared the ibMP levels in shaded and exposed Cabernet Franc vines from three different blocks at ten different time points (from five to 130 days post-bloom). While there seemed to be no significant differences in ibMP levels between the shaded and exposed clusters at harvest, pre-véraison light exposure was shown to effect the accumulation of ibMP in the berries (Ryona et al., 2008).

Scheiner et al. (2012) reported that vines with less water stress tended to be more vigorous

and bear fruit with higher ibMP levels. It appears that soils with a greater water-holding capacity (clay-rich soil) will favour vine growth and yield higher levels of ibMP in the grapes. Cabernet Sauvignon grapes grown on sandy-silt soil were reported to have higher ibMP levels than grapes from gravel soils (Roujou de Boubée et al., 2000).

Winemaking practices also affect ibMP levels. ibMP is easily extracted from crushed grape bunches at the beginning of pressing (Roujou de Boubée et al., 2002), and prolonged maceration on the skins in the presence of ethanol yields higher levels of ibMP (Kotseridis et al., 1999a).

Thermovinification reduces the ibMP levels; however, it is not a selective technique and it also removes desirable aroma compounds from the wine (Roujou de Boubée et al., 2002). Settling proved to be efficient for reducing ibMP levels (Roujou de Boubée et al., 2002).

Table 4: Aromatic properties and odour thresholds of the main methoxypyrazines that have been

detected in Cabernet Sauvignon wines.

Methoxypyrazines Aroma* Odour threshold

(ng/l)

Concentrations reported in Cabernet

Sauvignon wines

ipMP Green peas 1–21a 2–163

sbMP Green peas 1–21a _nf

ibMP Bell pepper 152b _2–243_/3,6–56,34

1 Maga & Sizer, 1973; 2 Roujou de Boubée et al., 2000; 3 Preston et al., 2008; 4 Allen et al., 1994 a In water; b In red wine

nf= not found

3.2 C6 and C9 derivatives: Green leaf volatiles

Short-chain aldehydes and alcohols such as trans-2-hexenal, cis-3-hexenol, 1-hexanol and nona-2,6-dienal are formed from the dioxygenation of linoleic acid (C18:2) and linolenic acid

(C18:3) in the lipoxygenase pathway. The C6 and C9 derivatives play an important role in plants,

as they are involved in wound healing and pest resistance or have antimicrobial and antifungal activity: The conversion of linolenic acid and linoleic acid to short-chain volatiles is activated by cell membrane disruption caused by crushing. These alcohols and aldehydes are characterised by a fresh green odour and can cause leafy-grassy off-odours in wine (Joslin & Ough, 1978; Schwab et al., 2008).

C6 and C9 derivatives are produced in the berry and evolve from aldehydes to alcohols in

the period from véraison to maturity (Kalua & Boss, 2009). Canuti et al. (2009) reported significant concentrations of hexanal, trans-2-nonenal and trans-2-hexenal in grape berries, but only trans-2-hexenal was found in the corresponding wines at levels much lower than its odour threshold. The rapid extraction and degradation or loss of trans-2-hexenal associated with an increase in the levels of 1-hexanol during fermentation, reported by Callejón et al. (2012), is in agreement with the rapid reduction of trans-2-hexenal to hexanol during fermentation, as described by Joslin and Ough (1978). The reduction of aldehydes to alcohols during fermentation has a positive impact on wine flavour, as alcohols have higher odour thresholds than their aldehyde counterparts and also have the potential to be converted into esters, which

contribute fruity notes. Some of the main C6 derivatives that have been detected in Cabernet

Table 5: Main C6 derivatives that have been detected in Cabernet Sauvignon wines. Aldehydes/alcohols

C6 derivatives

Aroma Odour threshold

a

(µg/l)

Concentrations reported in Cabernet Sauvignon wines

hexanol Green, cut grass 8 0001a 11 382–28 4202

Cis-3-hexenol Powerful, fresh green,

grass odour 400

1a _{706–1 522}2 _{/ 6.1-27.3}3

Trans 2-hexenol Green, citrusy,

orange, pungent odour 400

2b _151–7532

1 Guth 1997; 2 Tao & Zhang, 2010; 3 Bindon et al., 2013b a In water/ethanol (90+10,w/w); b In model wine

3.3 Monoterpenes

Monoterpenes are a grape-derived class of compounds that generally contribute to floral and citrus characters in wines. Terpenes are present in the grape skin, and their levels increase during grape maturation. Red varieties are not characterised by high levels of terpenes (Robinson et al., 2014).

The eucalyptus and mint aroma attributes that often characterise Cabernet Sauvignon wines have been positively correlated with 1,8-cineole, otherwise known as eucalyptol, and hydroxyl citronellol (Robinson et al., 2011). 1,8-Cineole is described as fresh, cool, medicinal and camphoraceous. The perception and recognition thresholds in a Californian Merlot wine were 1.1 and 3.2 µg/l respectively (Capone et al., 2011). 1,8-Cineole is produced in Cabernet Sauvignon grapes during berry development, although levels decrease during ripening and cannot contribute significantly to wine aroma (Kalua & Boss, 2009).

A survey of 190 commercial Australian red and white wines showed that only red varieties exhibited significant levels of 1,8-cineole (Capone et al., 2011). The same study reported that an increase in 1,8-cineole occurs during fermentation, but this stops once the skins are removed, indicating that the compound is extracted from grape skins. The proximity of Eucalyptus trees to grapevines can directly influence the concentration of 1,8-cineole in the corresponding wines. It was also shown that 1,8-cineole levels are generally highest in grapevine leaves, followed by the stems and then the grapes (Capone et al., 2012).

4

Chemical composition and sensory analysis of Cabernet Sauvignon wines

Several hundred volatile compounds contribute to the overall perceived aroma properties of wine. Gas chromatography techniques still play an important role in the identification and quantification of volatile compounds, but alone do not necessarily provide information on the perceptual properties of the detectable compounds. The introduction of chromatographic analyses coupled with olfactometric detection has enabled researchers to evaluate the odour intensities of the volatile compounds present in wines. In this technique, the sensory properties of the volatile compounds separated by gas chromatography are evaluated by a trained panel (Polášková, et al., 2008; Ebeler & Thorngate, 2009).

Typically, impact odorants have high odour intensities and low odour thresholds (low ppb or low ppt levels). Gas chromatography-olfactometry (GC-O), coupled with gas chromatography- mass spectrometry (GC-MS), thus is very useful to identify and quantify active or impact odorants at trace levels (Polášková, et al., 2008). A major limitation of GC-O techniques, however, is that they only evaluate the contribution of individual aroma volatiles, not taking into account the additive or suppressive effects that may occur between different compounds. Complex chemical interactions that are not always well understood come into play, expressing suppressing/masking or enhancing/additive effects. Impact odorants at above thresholds concentrations can have suppressive effects, whilst a group of compounds present at below threshold concentrations will have an enhancing effect and contribute to a specific aroma attribute perceived in the wine (Polášková, et al., 2008).

Sensory analyses, and particularly descriptive analysis, have been used extensively in combination with chemical analyses to determine the intensities of sensory attributes and how the volatile compounds are perceived in a given set of samples. Statistical modelling procedures, such as principal component analysis (PCA), partial least squares (PLS) and multiple factor analysis (MFA) are applied to sensory and chemical data and provide valuable information on how active odorants are positively or negatively correlated with certain aroma attributes. (Noble & Ebeler, 2002; Francis & Newton, 2005). Omission or addition tests in reconstituted extracts are useful to characterise impact odorants and confirm potential additive or suppressive interactions between compounds (Escudero et al., 2007; Francis & Newton, 2005; Plutowska & Wardencki, 2008).

As a red grape cultivar grown to produce some of the most prestigious and expensive wines worldwide, Cabernet Sauvignon has been the subject of extensive research. In the past 20 years, several studies using GC-O techniques combined with GC-MS have been conducted to characterise impact components in Cabernet Sauvignon wines (Lopez et al., 1999; Kotseridis & Baumes, 2000; Gȕrbȕz et al., 2006; Falcao et al., 2008). In these studies, 100+ compounds are detected and identified by matching their linear retention index (LRI) values with their corresponding aroma descriptors. There often are significant differences in the number and type of active components observed in the different studies due to differences in sample preparation

(Gȕrbȕz et al., 2006; Plutowska & Wardencki, 2008). Nonetheless, a number of volatile compounds have repeatedly been reported as being active odorants in Cabernet Sauvignon wines, as listed in Table 6.

Table 6: Active odorants identified in Cabernet Sauvignon wines.

Active odorants Odour threshold (µg/l) Concentrations reported in Cabernet Sauvignon wines Ethyl hexanoate 4401a 1 000-1 4009/373-1 31510 Ethyl octanoate 9601a _125-74110 Ethyl butyrate 6001a _{530-1 941}10 Isoamyl acetate 18301a 205-2 78410 Isoamyl alcohol 30 0003c 179 000–205 0009/30 783–140 08610 -ionone 0.094b _0.196-0.3724 -damascenone 2-75e _3-2110 2-phenylethanol 140002b 30 800–140 10010 Dimethylsulphide 10-1606d 5–6011 3-isobutyl-2-methoxypyrazine 0.0157e _0–547 Eucalyptol 1.18e 0,18–288 Hexanol 8 0003c 11 382–28 42010 Cis-3-hexenol 4003c 706–1 52210

1 Pineau et al., 2009; 2 Ferreira et al., 2000; 3 Guth 1997; 4 Kotseridis et al., 1999b; 5 Pineau et al., 2007; 6 Mestres

et al., 2000; 7 Roujou de Boubée et al., 2000; 8 Capone et al., 2011; 9 Bindon et al., 2013b; 10 Tao & Zhang, 2010;

11 Park et al., 1994

When studies also included a descriptive sensory analysis of the Cabernet Sauvignon wines that had been analysed chemically, two sets of sensory attributes often emerged: either fruity/berry-like or herbaceous/vegetative (Kotseridis et al., 2000; Chapman et al., 2005; Falcao et al., 2008; Preston et al., 2008; Robinson et al., 2011; Bindon et al., 2013a).

Studies have been conducted recently to establish relationships between the sensory attributes and chemical composition of Cabernet Sauvignon wines (Preston et al., 2008; Robinson et al., 2011; Bindon et al., 2013a; Hjelmeland et al., 2013; Bindon et al., 2014). In a study published in 2008, Preston et al. focused on the vegetal aroma characteristics of 16 selected Californian Cabernet Sauvignon wines. A descriptive analysis was conducted and the results were compared to the levels of ipMP and ibMP in the wines. It should be noted that only four of the 16 wines presented levels of ibMP above threshold, and the highest level of ibMP found in those wines was 24 ng/l. The concentrations of the pyrazines alone did not correlate well with any of the sensory attributes, indicating that other volatiles also affected the vegetal character of the wines.

In 2011, Robinson et al. studied 30 Cabernet Sauvignon wines (24 from Australian regions, three from Bordeaux and three from the Napa Valley). Three hundred and three volatile compounds were significantly different among the wines, and 232 of these were common to all 30 wines. The statistical analyses of the sensory attribute data with the chemical composition showed a clear distribution of the wines according to fruity and vegetal/herbaceous characteristics. The bell pepper attribute was positively correlated with ibMP and negatively correlated with δ octalactone, γ octalactone, γ decalactone and vitispirane. The red berry and dried fruit aroma attributes were positively correlated with ethyl and acetate esters.

In 2013, Hjelmeland et al. studied a total of 24 wines from different vintages and regions in California and Washington State, including 14 monovarietal Cabernet Sauvignon and 10 Bordeaux blends with Cabernet Sauvignon as a main component. The wines were selected based on interest from wine companies and to represent either fruity or vegetal sensory properties. Sixty-one targeted analytes were measured and only 56 were detected. The chemical composition was compared to a descriptive sensory analysis to determine whether chemical analyses could predict sensory profiles. The wines were differentiated in part as a result of varying alcohol levels. Thirty-six of the 56 detected compounds contributed significantly to the prediction of the sensory attributes. These compounds included hexyl acetate, ethyl octanoate, isobutanol, isoamyl alcohol, 2-phenylethanol, -ionone and linalool. Berry aroma was positively associated with hexyl acetate. Vegetal aroma was negatively associated with ethyl isobutyrate, isobutanol and 2-phenylethanol and positively correlated with ibMP and eucalyptol, although these two compounds did not present a strong, significant correlation with this attribute.

In most of the studies, some attributes were poorly explained by the volatile compounds measured, and some volatile compounds did not correlate well with the attributes. In particular,

ibMP often fails to correlate with the vegetative/herbaceous attributes, especially because most wines analysed presented below-threshold concentrations of ibMP (Preston et al., 2008; Bindon et al., 2013b; Hjelmeland et al., 2013). Although 3MH and 3MHA have been described as contributing to the aromatic complexity of Cabernet Sauvignon wines (Bouchilloux et al., 1998), none of the studies cited earlier measured these compounds or could report on their levels and their impact on the aromatic profiles of the respective wines studied (Robinson et al., 2011; Hjelmeland et al., 2013; Bindon et al., 2014).

5 Conclusion

Cabernet Sauvignon wines are often differentiated by two antagonistic aromatic profiles: one with fruity, berry-like aromas and the other one with vegetative, herbaceous aromas. The aroma compounds responsible for these two independent profiles are derived directly or indirectly from yeast and bacterial metabolism, and are determined by the grape composition at the time of harvest (Swiegers et al., 2005; Carey et al., 2008). Advances in analytical techniques have helped identify some of the impact odorants responsible for these typical characters. Thus, the measurement of a selected and limited number of volatile compounds combined with descriptive sensory analysis can help predict the sensory profiles of Cabernet Sauvignon wines (Hjelmeland et al., 2013; Bindon et al., 2014).

To our knowledge, no such study has been conducted on South African Cabernet Sauvignon wines yet. Bearing in mind that Cabernet Sauvignon is grown to produce some of the most expensive wines, the South African wine industry may gain valuable information from understanding the impact of volatile compounds on the sensory properties of their wines. This information could be used to make decisions at the viticultural and winemaking level to produce Cabernet Sauvignon wines with more desirable sensory attributes (Francis & Newton, 2005; Forde et al., 2011).

6 References

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